Production of glycoproteins using manganese

ABSTRACT

Culture media comprising manganese and methods of culturing cells to improve sialylation and glycosylation of glycoproteins are provided.

This application is a continuation of and claims priority to U.S. patentapplication Ser. No. 15/004,716, filed Jan. 22, 2016, currently pending,which is a continuation of and claims priority to U.S. patentapplication Ser. No. 14/087,747, filed Nov. 22, 2013, now U.S. Pat. No.9,273,110, which is a continuation of U.S. patent application Ser. No.13/112,940, filed May 20, 2011, now U.S. Pat. No. 8,617,878, which is acontinuation of U.S. patent application Ser. No. 11/634,506, filed Dec.6, 2006, now U.S. Pat. No. 7,972,810, which claims the benefit of U.S.Patent Application No. 60/748,880, which was filed Dec. 8, 2005, all ofwhich are hereby incorporated by reference in their entirety.

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a sequence listing in“txt” format and is identified by the file name: A-1030-US-CNT-4SeqListFromParent-MGB120106.txt, created Dec. 1, 2006, which is 6 KB insize. The subject matter contained in the electronic format of thissequence listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to cell culturing methods and media containingmanganese that improve glycosylation or sialylation of glycoproteins,including erythropoietin and analogs or derivatives thereof.

BACKGROUND

Erythropoietin (EPO) is a glycoprotein hormone that is normallysynthesized and secreted by peritubular cells in the kidney andfunctions as the principle homeostatic regulator of red blood cellproduction. Recombinant human erythropoietin (rHuEPO) is used clinicallyto treat anemias and increase red blood cell production in numerousdifferent conditions, such as perisurgery, chronic renal failure, sideeffects of HIV or HCV treatment, and side effects of cancerchemotherapy. Pharmaceutical biosynthesis of glycoproteins such as EPOis complicated by the need for both high levels of expression andappropriate posttranslational processing, which involves the addition ofN-linked and 0-linked branched oligosaccharide chains.

In glycoproteins, sugars are attached either to the amide nitrogen atomin the side chain of asparagine (termed an N-linkage) or to the oxygenatom in the side chain of serine or threonine (termed an O-linkage). Theprocess for forming N-linked carbohydrates begins with the addition of14 monosaccharides to a lipid-linked dichol in the endoplasmic reticulum(ER). After its formation, this carbohydrate complex is then transferredto the protein by the oligosaccharyltransferase (OST) complex in aprocess termed “core glycosylation” in the ER. Theoligosaccharyltransferase (OST) complex is a multi-protein unitcomprised of ribophorin I, II, OST48 and DAD1 (Kelleher and Gilmore 1997PNAS 94(10):4994-4999; Kelleher et al. 2003 Molecular Cell12(1):101-111; Kelleher et al. 1992 Cell 69(1):55-65).

Subsequently, the polypeptides are transported to the Golgi complex,where the O-linked sugar chains are added and the N-linked sugar chainsare modified in many different ways. In the cis and medial compartmentsof the Golgi complex, the original 14-saccharide N-linked complex may betrimmed through removal of mannose (Man) residues and elongated throughaddition of N-acetylglucosamine (GlcNac) and/or fucose (Fuc) residues.The various forms of N-linked carbohydrates have in common apentasaccharide core consisting of three mannose and twoN-acetylglucosamine residues. Finally, in the trans Golgi, other GlcNacresidues can be added, followed by galactose (Gal) and a terminal sialicacid (Sial). Carbohydrate processing in the Golgi complex is called“terminal glycosylation” to distinguish it from core glycosylation.

Sialic acid is a generic name for a family of about 30 naturallyoccurring acidic monosaccharides that are frequently the terminal sugarsof carbohydrates found on glycoproteins and glycolipids. Sialylation ofrecombinant glycoproteins is very important and may impart manysignificant properties to the glycoprotein including charge,immunogenicity, resistance to protease degradation, plasma clearancerate, and bioactivity.

The final complex carbohydrate units can take on many forms, some ofwhich have two, three or four branches (termed biantennary, triantennaryor tetraantennary). An exemplary N-linked biantennary structure is shownbelow:

Man-GlcNac-Gal-Sial/Asparagine-GlcNac-GlcNac-Man\Man-GlcNac-Gal-Sial

A number of enzymes involved in glycosylation utilize divalent cationsas co-factors. For example, numerous enzymes involved in thedolichol-linked oligosaccharide synthesis require divalent cations asco-factors for activity (Couto et al. 1984 J. Biol. Chem.259(1):378-382; Jensen and Schutzbach 1981 J. Biol. Chem.256(24):12899-12904; Sharma et al. 1982 European Journal of Biochemistry126(2):319-25). The enzyme which catalyses the addition of 0-linkedcarbohydrate to the polypeptide also requires a divalent cation foractivity (Sugiura et al. 1982 J. Biol. Chem. 257(16):9501-9507).Manganese (Mn++) is a required co-factor for the enzymeβ-galactoside-α-1,3,-galactosyltransferase, which catalyzes the additionof terminal galactose to elongating N-acetyl-glucosamine sugars (Witsellet al. 1990 J. Biol. Chem. 265(26):15731-7). It was previously reportedthat manganese at a concentration of 0.1 mM or 1 mM partially reversedthe reduction in N-linked and O-linked occupancy of erythropoietincaused by A23187, a compound which depletes divalent cations (Kaufman etal. 1994 Biochemistry 33(33):9813-9).

rHuEPO has previously been shown to contain three N-linked and oneO-linked branched carbohydrate structures that are highly sialylated(Takeuchi et al. 1988 J. Biol. Chem. 263(8):3657-3663). De-sialylatedEPO is virtually inactive to induce erythropoiesis in vivo due to therapid clearance of this modified protein by the hepatocyte asialoglycoprotein receptor (Ashwell and Harford 1982 Annual Review ofBiochemistry 51(1):531-554; Goochee et al. 1991 Bio/Technology.9(12):1347-55). Other studies have shown that sialylation andglycosylation decreases binding kinetics of EPO to the EPO receptor.(Darling et al. 2002 Biochemistry 41(49):14524-31.)

Darbepoetin alfa is a novel glycosylation analog of recombinant humanerythropoietin (rHuEPO) that contains two additional N-linkedglycosylation sites. Darbepoetin has decreased receptor-binding activitybut exhibits a three-fold longer serum half-life and increased in vivoactivity as a result of this increased persistence in circulation. Thein vivo activity of EPO analogs has been demonstrated to correlate withthe number of N-linked carbohydrates. (Elliott et al., Exp Hematol. 200432(12):1146-55.)

rHuEPO produced in CHO cells can exhibit a variable extent ofglycosylation and sialylation. (Takeuchi et al., 1989 PNAS86(20):7819-22, Zanette et al., 2003 Journal of Biotechnology101(3):275-287). Given that EPO sialylation is an important factor in invivo bioactivity, consistency in glycosylation and higher levels ofsialylation of rHuEPO and its analogs are desirable qualities whenproducing recombinant protein for therapeutic uses. Thus, there exists aneed for culture media and culturing methods that improve theglycosylation or sialylation of glycoproteins produced in cell cultures.

SUMMARY OF THE INVENTION

In one aspect, the invention provides culture media comprising hostcells and a non-toxic amount of manganese effective to increase thesialylation of a glycoprotein composition produced by such host cells.

In another aspect, the invention provides methods for improvingsialylation of glycoproteins by growing host cells producing suchglycoproteins in a culture medium containing manganese, in an amounteffective to increase the sialylation of such glycoproteins.

Exemplary glycoproteins include erythropoiesis-stimulating molecules,such as erythropoietin and darbepoetin. The manganese may be present inan amount effective to increase sialylation, either through increasingthe percentage of sialylated molecules produced or through increasingtheir degree of sialylation, and/or effective to increase occupancy ofO-linked or N-linked glycosylation sites, and/or effective to increasegalactosylation. Preferably the addition of manganese to culture mediumimproves such a property(ies) by at least 10%, 20%, 30%, 40%, 50%, 60%,70%, or more relative to culture media lacking manganese or culturemedia containing a lower concentration of manganese.

In one exemplary embodiment, the invention provides a method forproducing an erythropoietic composition comprising sialylatederythropoiesis-stimulating molecules, wherein the method involves thestep of growing a manganese-responsive host cell in culture mediumcontaining manganese, and optionally includes the step of recovering anerythropoietic composition characterized by any one, two, three, four ormore of the following improved properties: (1) a reduced percentage of“lower sialylated” erythropoiesis-stimulating molecules, e.g. less thanabout 5% of the molecules are lower sialylated; (2) an increasedpercentage of “highly sialylated” erythropoiesis-stimulating molecules;(3) an increased percentage of erythropoiesis-stimulating moleculeswhich are glycosylated at potential O-linked glycosylation sites; (4) anincreased percentage of galactose among the sugars attached toerythropoiesis-stimulating molecules, or (5) an increased percentageoccupancy of potential N-linked glycosylation sites.

The manganese in the culture medium is at a concentration that iseffective to provide one or more of such improved properties, e.g.ranging from about 0.01 to about 40 μM, from about 0.1 to about 10 μM,or from about 0.4 to about 4 μM.

In any of the preceding culture media or methods, the culture medium maybe essentially serum-free and/or may optionally comprises one or moresupplementary amino acids selected from the group consisting ofasparagine, aspartic acid, cysteine, cystine, isoleucine, leucine,tryptophan, or valine.

The host cell may be any mammalian cell, e.g. a CHO cell, and may begrown in any suitable culture system, e.g. in roller bottles.

The manganese may be present in the initial growth medium or may beadded after a rapid cell growth phase, e.g. a period ranging betweenabout 2 and 20 days, or may be added after one or two harvest cycles.

Other features and advantages of the invention will become apparent fromthe following detailed description. It should be understood, however,that the detailed description and the specific examples, whileindicating exemplary embodiments of the invention, are given by way ofillustration only, because various changes and modifications within thespirit and scope of the invention will become apparent to those skilledin the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 displays the amount of rHuEPO in the flow through fraction as apercentage of the amount loaded onto the column and shows results fromculture medium with no added manganese, and with 4 μM added manganese.

FIG. 2 displays the amount of rHuEPO in the IEX-retained fraction as apercentage of the amount loaded onto the column and shows results fromculture medium with no added manganese, and with 4 μM added manganese.

FIG. 3 displays the amount of darbepoetin in the IEX-retained fractionas a percentage of the amount loaded onto the column, after each harvestcycle, and shows results from culture medium with no added manganese,and with 4 μM added manganese.

FIG. 4 displays percent of rHuEPO molecules in which O-sites wereoccupied with glycosylation and shows results from culture medium withno added manganese, and with 4 μM added manganese.

FIG. 5 displays percent of darbepoetin molecules in which O-sites wereoccupied with glycosylation, after each harvest cycle, and shows resultsfrom culture medium with no added manganese, and with 4 μM addedmanganese.

FIG. 6 displays percent of darbepoetin molecules in which O-sites wereoccupied with glycosylation and shows results from culture medium withvarying concentrations of manganese.

FIG. 7 shows representative glycosylation forms identified by MALDI-TOFMS.

FIG. 8 displays percent of recoverable highly sialylated rHuEPO obtainedafter culturing in Standard Media, Enriched Media (Standard Mediasupplemented with amino acids), and Enriched Media with varyingconcentrations of manganese.

FIG. 9 displays the percent of lower sialylated rHuEPO forms obtainedafter culturing in Standard Media, Enriched Media, and Enriched Mediawith varying concentrations of manganese.

FIG. 10 displays percentage of O-site occupancy by glycosylationobtained after culturing in Standard Media, Enriched Media, and EnrichedMedia with varying concentrations of manganese.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides culture media and cell culture methods thatimprove the sialylation of glycoproteins, particularlyerythropoiesis-stimulating molecules such as erythropoietin of SEQ IDNO: 3, or analogs, variants, or derivatives thereof, includingdarbepoetin of SEQ ID NO: 2.

Recombinant glycoproteins produced in CHO cells can exhibit variableglycosylation and sialylation. Highly sialylated forms of glycoproteinmolecules can be separated from lower sialylated (includingnon-sialylated) forms of such molecules via anion exchangechromatography. Sialic acids, being acidic and thus negatively charged,are captured on the column, so that highly sialylated molecules areretained on the column while lower sialylated forms flow through. Theamount of glycoprotein in each fraction (retained on column vs. flowthrough fraction) can be determined and compared to the starting amountof glycoprotein loaded from the cell culture media.

The addition of manganese to culture medium has been shown herein toresult in significant alterations in post-translational processing oferythropoiesis-stimulating molecules, such as erythropoietin anddarbepoetin, by the cultured cells producing erythropoietin. Manganesedecreases the amount of lower sialylated glycoprotein produced (andincreases the amount of highly sialylated glycoprotein recovered),increases the number of potential O-linked glycosylation sites that areoccupied by sugar chains, increases the number of potential N-linkedglycosylation sites that are occupied by sugar chains, increases theterminal galactosylation of sugar chains, and increases terminalsialylation of sugar chains. Manganese did not appear to alter thedegree of branching of the sugar chains (e.g. one, two, three or fourbranches). Manganese also reverses the reduction in sialylation observedwhen the culture medium is periodically supplemented with amino acidsdepleted during cell culture, e.g. asparagine, aspartic acid, cysteine,cystine, isoleucine, leucine, tryptophan, and valine.

The term “erythropoietic composition” as used herein means a collectionof erythropoiesis stimulating molecules that contain glycosylationsites, and among which at least some of the molecules carry a sugarchain comprising at least one terminal sialic residue (i.e. suchmolecules are “sialylated”). Similarly, the term “glycoproteincomposition” as used herein means a collection of glycoproteinmolecules, among which at least some of the molecules are sialylated.

The term “erythropoiesis-stimulating molecules” as used herein includeshuman erythropoietin (SEQ ID NO.: 3) or a biologically active variant,derivative, or analog thereof, including a chemically modifiedderivative of such protein or analog. Amino acids 1 through 165 of SEQID NO: 3 constitute the mature protein. Another exemplaryerythropoiesis-stimulating molecule is darbepoetin (SEQ ID NO: 2). Aminoacids 1 through 165 of SEQ ID NO: 2 constitute the mature protein. Alsocontemplated are analogs of erythropoietin (SEQ ID NO.: 3) ordarbepoetin (SEQ ID NO: 2), with 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 3 or SEQ IDNO: 2, respectively, and still retaining erythropoietic activity.

Exemplary sequences, manufacture, purification and use of recombinanthuman erythropoietin are described in a number of patent publications,including but not limited to Lin U.S. Pat. No. 4,703,008 and Lai et al.U.S. Pat. No. 4,667,016, each of which is incorporated herein byreference in its entirety. Darbepoetin is a hyperglycosylatederythropoietin analog having five changes in the amino acid sequence ofrHuEPO which provide for two additional carbohydrate chains. Morespecifically, darbepoetin contains two additional N-linked carbohydratechains at amino acid residues 30 and 88 of SEQ ID. NO: 2. Exemplarysequences, manufacture, purification and use of darbepoetin and othererythropoietin analogs are described in a number of patent publications,including Strickland et al., 91/05867, Elliott et al., WO 95/05465,Egrie et al., WO 00/24893, and Egrie et al. WO 01/81405, each of whichis incorporated herein by reference in its entirety.

As used herein, “analogs” refers to an amino acid sequence that hasinsertions, deletions or substitutions relative to the parent sequence,while still substantially maintaining the biological activity of theparent sequence, as determined using biological assays known to one ofskill in the art. “Variants” include naturally occurring allelicvariants, splice variants, or polymorphic forms of the parent sequence.“Derivatives” of naturally occurring, variant or analog polypeptidesinclude those which have been chemically modified, for example, toattach water soluble polymers (e.g., polyethylene glycol),radionuclides, or other diagnostic or targeting or therapeutic moieties,any of which can be attached directly or indirectly through linkers.

The term “erythropoietic activity” means activity to stimulateerythropoiesis as demonstrated in an in vivo assay, for example, theexhypoxic polycythermic mouse assay. See, e.g., Cotes and Bangham,Nature, 191:1065 (1961).

The term “manganese-responsive host cell” as used herein means a hostcell which produces a glycoprotein and which responds to added manganesein its culture medium by increasing sialylation, either by increasingthe percentage of sialylated glycoprotein molecules produced or byincreasing the degree of sialylation (i.e. the number of sialic acidsper molecule) of the glycoprotein molecules produced. For erythropoieticcompositions, manganese-responsive host cells include host cells thatrespond to added manganese by increasing the percentage of highlysialylated erythropoiesis-stimulating molecules recovered after anionexchange chromatography carried out as described below. In exemplaryembodiments, manganese-responsive host cells may include host cellsgrowing anchored to a solid surface, e.g. in roller bottles. Anymanganese-responsive host cells described herein may be used accordingto the invention.

Culture Medium Components

The invention provides a culture medium comprising an amount ofmanganese effective to increase the sialylation of a glycoproteincomposition produced by cells grown in this culture medium. In oneembodiment, said amount of manganese is non-toxic to the cells, i.e.,does not reduce cell viability, cell growth or protein production. Inrelated embodiments, the invention provides a culture medium comprisingan amount of manganese effective to increase the sialylation of anerythropoietic composition produced by cells grown in this culturemedium.

The amount of manganese in the culture media of the invention may begreater than the “trace element” amount present in standard mediacompositions, e.g., greater than 0.001 μM. While the quality oferythropoietic compositions is clearly improved by the addition of 40 μMmanganese to host cell cultures, in some cases the yield of proteinsecreted into the media is substantially reduced, indicating a toxiceffect of such a concentration of manganese. In exemplary embodiments,the concentration of manganese in the culture medium (i.e. the finalconcentration after the manganese-supplemented medium is added to thehost cells in culture) ranges from about 0.01 to about 40 μM, or fromabout 0.1 to about 10 μM, or from about 0.4 to about 4 μM. In otherexemplary embodiments, the concentration of manganese at the lower endof the desired range may range from about 0.005, 0.01, 0.05, 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5 or 4 μM orhigher; the concentration of manganese at the higher end of the rangemay also range up to about 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7,6, 5 or 4 μM.

The concentration of manganese in the culture medium added to the cellsmay be adjusted to achieve the desired final concentration of manganesein the culture system. For example, with batch processes involvingcomplete removal and replacement of culture medium, replacement culturemedium containing 4 μM Mn²⁺ is added to the cells to achieve a finalculture medium at 4 μM Mn²⁺. Alternatively, when continuous perfusionprocesses are used, the concentration of manganese in the added mediawill need to be higher to achieve a final culture medium at the desiredMn²⁺ concentration within a range. Adjustment of the concentration canbe easily carried out by one of ordinary skill in the art.

The culture medium can also include any other necessary or desirableingredients known in the art, such as carbohydrates, including glucose,essential and/or non-essential amino acids, lipids and lipid precursors,nucleic acid precursors, vitamins, inorganic salts, trace elementsincluding rare metals, and/or cell growth factors. The culture mediummay be chemically defined or may include serum, plant hydrolysates, orother derived substances. The culture medium may be essentially orentirely serum-free or animal-component free. Essentially serum-freemeans that the medium lacks any serum or contains an insignificantamount of serum.

The culture medium may also include supplementary amino acids depletedduring cell culture, e.g. asparagine, aspartic acid, cysteine, cystine,isoleucine, leucine, tryptophan, and valine. The amino acidsupplementation may be in the initial growth medium and/or in mediumadded during or after the rapid growth phase.

The medium may include lipids and/or lipid precursors such as choline,ethanolamine, or phosphoethanolamine, cholesterol, fatty acids such asoleic acid, linoleic acid, linolenic acid, methyl esters,D-alpha-tocopherol, e.g. in acetate form, stearic acid; myristic acid,palmitic acid, palmitoleic acid; or arachidonic acid. A number ofcommercially available lipid mixtures are available.

The medium may include an iron supplement comprising iron and asynthetic transport molecule to which the iron binds. The medium mayinclude inorganic compounds or trace elements, supplied as appropriatesalts, such as sodium, calcium, potassium, magnesium, copper, iron,zinc, selenium, molybdenum, vanadium, manganese, nickel, silicon, tin,aluminum, barium, cadmium, chromium, cobalt, germanium, potassium,silver, rubidium, zirconium, fluoride, bromide, iodide and chloride. Anumber of commercially available mixtures of trace elements areavailable.

The medium may also optionally include a nonionic surfactant orsurface-active agent to protect the cells from the mixing or aeration.The culture medium may also comprise buffers such as sodium bicarbonate,monobasic and dibasic phosphates, HEPES and/or Tris.

In exemplary embodiments, the media is DMEM/F-12 media (Gibco)containing 5% serum. DMEM includes the following inorganic salts:Calcium Chloride, Cupric sulfate Ferric-Nitrate or Sulfate, PotassiumChloride, Magnesium Sulfate or Chloride, Sodium Chloride, SodiumDihydrogen Phosphate, Sodium Bicarbonate, Zinc sulfate; the followingamino acids L-Alanine, L-Arginine, L-Asparagine, L-Aspartic acid,L-Cysteine, L-Glutamic acid, L-Glutamine, Glycine, L-Histidine,L-Isoleucine, L-Leucine, L-Lysine, L-Methionine, L-Phenylalanine,L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine, L-Valine;the following lipids and vitamins: Biotin, D-Calcium-Pantothenate,Choline Chloride, Folic Acid, myo-Inositol, Niacinamide, Nicotinamide,Pyridoxine, Riboflavin, Thiamine, Vitamin B12 (cobalamin), Thymidine,Linoleic Acid, Lipoic Acid; and other components including D-Glucose,Phenol Red, Hypoxanthine, Sodium Pyruvate, Putrescine, and HEPES.

The culture medium may also comprise inducers of protein production,such as sodium butyrate, or caffeine. Other known inducers include, butare not limited to, the following compounds: N-Acetyl-L-cysteine,Actinomycin D, 7-Amino-, Bafilamycin A1, Streptomyces griseus,Calphostin C, Cladosporium cladosporioides, Camptothecin, Camptothecaacuminata, CAPE, 2-Chloro-2′-deoxyadenosine, 2-Chloro-2′-deoxyadenosine5′-Triphosphate, Tetralithium Salt, Cycloheximide, CyclophosphamideMonohydrate, Cyclosporine, Trichoderma polysporum, Daunorubicin,Hydrochloride, Dexamethasone, Doxorubicin, Hydrochloride,(−)-Epigallocatechin Gallate, Etoposide, Etoposide Phosphate,ET-18-OCH3, 5-Fluorouracil, H-7, Dihydrochloride, Genistein,4-Hydroxynonenal, 4-Hydroxyphenylretinamide, Hydroxyurea, IL-1βInhibitor, (±)-S-Nitroso-N-acetylpenicillamine, S-Nitrosoglutathione,Phorbol-12-myristate-13-acetate, Puromycin, Dihydrochloride,1-Pyrrolidinecarbodithioic Acid, Ammonium Salt, Quercetin, Dihydrate,Rapamycin, Sodium Butyrate, Sodium 4-Phenylbutyrate,D-erythro-Sphingosine, N-Acetyl-, D-erythro-Sphingosine, N-Octanoyl-,Staurosporine, Streptomyces sp., Sulindac, Thapsigargin, TRAIL, E. coli,Trichostatin A, Streptomyces sp., (±)-Verapamil, Hydrochloride,Veratridine, Vitamin D3, la, 25-Dihydroxy-, and Vitamin E Succinate (VWRand Calbiochem).

The culture medium optionally excludes A23187 or other compounds whichdeplete divalent cations.

Culturing Methods

The invention also provides methods for producing a glycoproteincomposition, such as an erythropoietic composition, which may includeculturing a manganese-responsive host cell in any of the culture mediadescribed herein. Such methods may further include the step ofrecovering the glycoprotein composition, e.g. the erythropoieticcomposition, from the host cells or culture medium. Manganese may beincluded in the initial culture medium during the initial growth phaseor may be added at later stages. Manganese may have a greater effectwhen added after a rapid growth phase during which maximum or nearmaximum host cell growth is achieved, for example, a period longer than2, 3, 4, 5, 7, 10, 15 or 22 days, or up to 22, 25, 30, 35, 40, 45, 50,or 55 days and may have an even greater effect after prolonged cellgrowth, e.g. after two harvest cycles. When the recombinant protein issecreted into the medium, the medium can be harvested periodically, sothat the same host cells can be used through several harvest cycles. Inexemplary embodiments, host cells producing erythropoiesis-stimulatingmolecules are incubated in three discrete batch harvest cycles. For eachcycle, medium is harvested and replaced with fresh medium. The firstcycle may be, e.g., 8 days; the second cycle, e.g., 7 days; and thethird cycle, e.g., 5 days in duration.

Any host cells known in the art to produce glycosylated proteins may beused, including yeast cells, plant cells, plants, insect cells, andmammalian cells. Exemplary yeast cells include Pichia, e.g. P. pastoris,and Saccharomyces e.g. S. cerevisiae, as well as Schizosaccharomycespombe, Kluyveromyces, K. Zactis, K. fragilis, K. bulgaricus, K.wickeramii, K. waltii, K. drosophilarum, K. thernotolerans, and K.marxianus; K. yarrowia; Trichoderma reesia, Neurospora crassa,Schwanniomyces, Schwanniomyces occidentalis, Neurospora, Penicillium,Totypocladium, Aspergillus, A. nidulans, A. niger, Hansenula, Candida,Kloeckera, Torulopsis, and Rhodotorula. Exemplary insect cells includeAutographa californica and Spodoptera frugiperda, and Drosophila.Exemplary mammalian cells include varieties of CHO, BHK, HEK-293, NS0,YB2/3, SP2/0, and human cells such as PER-C6 or HT1080, as well as VERO,HeLa, COS, MDCK, NIH3T3, Jurkat, Saos, PC-12, HCT 116, L929, Ltk-, WI38,CV1, TM4, W138, Hep G2, MMT, aleukemic cell line, embryonic stem cell orfertilized egg cell.

A variety of culture systems are known in the art, including T-flasks,spinner and shaker flasks, roller bottles and stirred-tank bioreactors.Roller bottle cultivation is generally carried out by seeding cells intoroller bottles that are partially filled (e.g., to 10-30% of capacity)with medium and slowly rotated, allowing cells to attach to the sides ofthe bottles and grow to confluency. The cell medium is harvested bydecanting the supernatant, which is replaced with fresh medium.Anchorage-dependent cells can also be cultivated on microcarrier, e.g.polymeric spheres, that are maintained in suspension in stirred-tankbioreactors. Alternatively, cells can be grown in single-cellsuspension.

Culture medium may be added in a batch process, e.g. where culturemedium is added once to the cells in a single batch, or in a fed batchprocess in which small batches of culture medium are periodically added.Medium can be harvested at the end of culture or several times duringculture. Continuously perfused production processes are also known inthe art, and involve continuous feeding of fresh medium into theculture, while the same volume is continuously withdrawn from thereactor. Perfused cultures generally achieve higher cell densities thanbatch cultures and can be maintained for weeks or months with repeatedharvests.

Methods for controlling sialylation of a recombinant glycoprotein,particularly for controlling N-glycolylneuraminic acid (NGNA) levels inthe sugar chains, are described in U.S. Pat. No. 5,459,031, incorporatedherein by reference in its entirety, and such methods may be used inconjunction with the culture media and culture methods described herein.The methods involve adjusting culture parameters, including the carbondioxide level, to achieve the desired NGNA content in carbohydrate.

Evaluation of Glycosylation and Sialylation

For glycoprotein compositions, an increase or improvement in sialylationcan be determined by anion exchange chromatography according to Elliottet al., Biochemistry, 33(37):11237-45 (1994), herein incorporated byreference in its entirety. More highly sialylated proteins are expectedto be more negatively charged and bind more strongly to the column,while less sialylated and asialoproteins flow through or are easilyeluted. The amount of glycoprotein molecules in each of the twofractions (retained on resin vs. flow through fraction) can bedetermined, e.g., by ELISA, and compared to the starting amount of suchmolecules loaded from the cell culture media. Exemplary ELISA kits aresold commercially and include R & D Systems, IVD Human EPO EIA kit.

Chromatography is carried out as follows. To eliminate cells and debris,medium in which mammalian cells that produce anerythropoiesis-stimulating molecule, or other glycoprotein, have beencultured is centrifuged at about 1000 rpm and filtered through a 0.45micron filter. This material is then subjected to anion exchangechromatography in order to prepurify a fraction containing primarily thefour to seven most highly sialylated species of the glycoproteinmolecules. A strong ion exchange resin may be used, such as, forexample, TRICORN™ Mono-Q 5/50 GL (Amersham, part #17-5166-01) or otherstrong anion exchange resins, particularly those that have thequaternary amine —CH₂—N—(CH₃)₃ as the functional group of the resin. Theexact procedure will depend on the theoretical maximum number of sialicacid residues that the particular glycoprotein molecules can contain.For example, a theoretical maximum number of sialic acid residues forhuman erythropoietin, which has 3 N-glycan sites and 1 O-glycan site, is(3×4)+2=14. This assumes that each N-glycan site can have up to fourbranches (since pentaantennary species are rare) and that each O-glycansite can have up to two branches. Making similar assumptions fordarbepoetin, which has 5 N-glycan sites and 1 O-glycan site, thetheoretical maximum for darbepoetin is (5×4)+2=22. The buffers used toelute the glycoprotein molecules from the anion exchange column aredesigned to: (1) elute from the column most or all protein moleculesbelonging to species that are less sialylated than a group of speciesconsisting of approximately the top third most highly sialylated species(for erythropoietin, the “highly sialylated” species are those having9-14 sialic acid residues per protein molecule, and for darbepoetin the“highly sialylated” species are those having 17-22 sialic acid residuesper protein molecule); (2) then elute protein molecules belonging to thefour to seven most highly sialylated species, and (3) finally removemore highly charged species from the column, which may includeglycoforms bearing sulfated N-glycans. Therefore, the exact compositionof the wash and elution buffers can be adjusted according to thetheoretical maximum number of sialic acid residues on the glycoproteinmolecule. One of skill in the art can make such adjustments based onroutine empirical optimization of column parameters and assaying thematerial coming off the column on analytical isoelectric focusing gels.

Analytical polyacrylamide isoelectric focusing gels that can separatedifferent charged forms of erythropoiesis-stimulating molecules bearingdifferent numbers of sialic acid residues can also be performedessentially as described in the Amersham-Pharmacia Guide to IsoelectricFocusing (APB, RW 5/5/98) in 6 M urea using commercially availableampholytes (pH 3 to 5 for human erythropoietin or pH 2 to 4 fordarbepoetin). Other pH ranges for ampholytes may be appropriate forother erythropoiesis-stimulating molecules with substantially differentnumbers of sialic acid residues.

For erythropoietin, the extent of sialylation is estimated bydetermining the percent of total erythropoetic protein loaded onto ananion exchange column that elutes in a fraction containing mostly highlysialylated species of erythropoietin having 9 to 14 sialic acid residuesper protein molecule.

For darbepoetin, the extent of sialylation is estimated by determiningthe percent of total erythropoetic protein loaded onto an anion exchangecolumn that elutes in a fraction containing mostly highly sialylatedspecies of darbepoetin having 18 to 22 sialic acid residues per proteinmolecule.

An increase in the percentage of erythropoiesis-stimulating moleculesrecovered from the pool retained on the resin (or a reduction in thepercentage of such molecules observed in the flow through fraction)relative to the control (e.g. produced from media with no manganese ortrace element amounts of manganese) indicates an increase insialylation, whether through increasing the percentage of sialylatedmolecules produced or through increasing their degree of sialylation.

The actual glycan structure can be determined by any techniques known inthe art, including enzymatic digestion of carbohydrate, lectinimmunoblotting, 1D and 2D 1H-NMR spectroscopy, mass spectroscopytechniques including electrospray ionization tandem mass spectrometry(ESI MS) or matrix assisted laser desorption ionization time-of-flightmass spectrometry (MALDI-TOF MS), and/or fluorescent labeling ofenzymatically released N-glycans followed by resolution by HPLC andcomparison to known N-glycan control samples.

An exemplary technique, described in the examples below, for determiningthe amount of glycoprotein with an occupied O-glycosylation siteinvolves N-Glycanase digestion to remove the N-linked carbohydratesfollowed by reverse phase-HPLC to separate the glycoprotein compositioninto two peaks. Peak identification as occupied O-site or unoccupiedO-site can be confirmed by mass spectrometry.

N-site branching and sialylation, including the percentage of sialylatedmolecules produced and the degree of sialylation of the sialyatedmolecules, can be determined by analyzing the glycoproteins forstructural content by N-glycan mapping and enzymatic sequencing, e.g. bydigestion with N-Glycanase and neuraminidase, coupled with MALDI-TOFmass spectrometry for size determination of the released sugars. Anexemplary technique is described in the examples below.

The percent of the sugars attached to the erythropoiesis-stimulatingmolecules that are galactose can be determined, e.g., by neuraminidaseplus galactosidase digestion followed by HPLC separation or MALDI-TOFmass spectrometry for size determination of the released sugars. Anexemplary technique is described in the examples below.

EXAMPLES Example 1

Protein Production Methods

This example describes a cell culture method for production ofrecombinant human erythropoietin (rHuEPO, SEQ ID NO: 3) or darbepoetin(SEQ ID NO: 2). A DHFR minus CHO cell line was stably transfected with agenomic DNA sequence containing the human erythropoietin gene (Lin U.S.Pat. No. 4,703,008, incorporated herein by reference) or a cDNA sequenceencoding darbepoetin gene (SEQ ID NO: 1). Roller bottles (850 cm²) wereinoculated with 1.7×10⁷ total cells and grown for 5 days in 450 mls of1:1 DMEM/F-12 media (Gibco) containing 5% serum. The cultures werewashed once with PBS and then incubated in three discrete batch harvestcycles. Media was replaced twice in the first 14 days; the third cyclewas 5 to 6 days in duration. For erythropoietin, at each harvest, theconditioned culture medium was completely removed and fresh 1:1DMEM/F-12 without serum (“Standard Media”) was added to replace theharvested media. When manganese was included in the culture medium,manganese chloride (Sigma) was added to all replacement culture media atthe desired concentration, e.g. 0.4, 4 or 40 μM. Roller bottles wereoverlaid with a gas mix containing 80 torr pCO₂, 130 torr pO₂, andbalanced N₂ after each media addition. Cells producing darbepoetin werecultured under conditions similar to cells producing erythropoietinexcept that the Standard Media was 2X 1:1 DMEM/F-12 (without serum).

Example 2

Quantitation of rHuEPO or Darbepoetin in Harvested Culture Media

200 μl of harvested media produced according to Example 1 was analyzedfor the amount of rHuEPO or darbepoetin produced, using reverse phaseHPLC. Samples were separated on a polymer PLRPS (4.6 mm×150 mm; 1000 Å(Polymer Laboratories) under reverse phase conditions (linear ABgradient from 30%-55% B over 17 minutes; buffer A: 0.1% TFA in H₂O,buffer B: 0.1% TFA in 90% CH₃CN (Sigma)). The retention time for rHuEPOor darbepoetin within the culture media was compared with a purifiedrHuEPO or darbepoetin standard (Amgen Inc.). Waters Millennium Softwarewas used to manually integrate the rHuEPO or darbepoetin peak areas toensure consistent integration. Integrated peak areas of unknown sampleswere quantitated by comparison to a known standard curve.

Example 3

Effect of Manganese on Highly Sialylated and Lower Sialylated Forms ofErythropoiesis-Stimulating Molecules

CHO cells producing rHuEPO were grown as described in Example 1 with andwithout added 4 μM MnCl₂. Conditioned media collected after the thirdharvest cycle was analyzed for percent recovery of highly sialylatedforms of rHuEPO. CHO cells producing darbepoetin were grown as describedin Example 1 with and without added 4 μM MnCl₂. Conditioned mediacollected after each of the three harvest cycles was analyzed.

Lower sialylated forms of rHuEPO were separated from highly sialylatedmaterial using anion exchange method as described in Elliott et al.,Biochemistry, 33(37):11237-45 (1994), herein incorporated by referencein its entirety. Briefly, highly sialylated rHuEPO, having a strongnegative charge, binds to the resin while lower sialylated rHuEPO washesthrough the column. Cell culture media from each roller bottle obtainedas described in Example 1 was first concentrated sixty-fold, to about5-15 mg/mL, using a 10,000 MWCO membrane then buffer exchanged into 10mM Tris pH 7.0. This concentrated and buffer exchanged media was loadedonto a strong anion exchange column having a quaternary amine as thefunctional group. Unbound material that flowed through at 10 mM Tris oreluted with low salt was collected as the lower sialylated fraction.Material bound to the ion exchange (IEX) column was eluted with highersalt as the “recoverable” highly sialylated fraction.

The total amount of rHuEPO in one or both fractions was measured usingan ELISA kit obtained from R&D Systems (Quantikine IVD Human EPO EIAkit), following the manufacturer's recommended procedure, and comparedto the total amount of rHuEPO in the harvest media loaded onto thecolumn. Dilutions of 1:1,000,000 and 1:500,000 of each flow-throughsample were made prior to analysis in order to fall within the standardcurve of the assay. A highly sialylated fraction of darbepoetin wasseparated using similar methods as described above to isolate theisoforms with 17-22 sialic acid residues and measured using RP-HPLC.

Results of a representative experiment for rHuEPO are shown in FIGS. 1and 2 . FIG. 1 displays the amount of rHuEPO in the flow throughfraction as a percentage of the amount loaded onto the column and showsthat the addition of 4 μM manganese reduced the lower sialylatedfraction of EPO compared to control, from 10.01% to 3.39%. FIG. 2displays the amount of rHuEPO in the IEX-retained fraction as apercentage of the amount loaded onto the column and shows that theaddition of 4 μM manganese increased the percent recoverable EPO in thehighly sialylated fraction compared to control, from 24% to 28%.

Results of a representative experiment for darbepoetin are shown in FIG.3 . FIG. 3 displays data from each harvest cycle and shows that theaddition of 4 μM manganese increased the percent recoverable darbepoetinin the highly sialylated fraction compared to control, from 32.8% to36.3% after the third harvest. The further addition of 7 mMN-acetylmannosamine appeared to provide a further increase in %recoverable darbepoetin to 41.1% after the third harvest.

These data demonstrate that addition of Mn²⁺ to culture medium decreasedproduction of lower sialylated forms of erythropoietin and darbepoetinin CHO cell cultures, and increased percent of recoverable highlysialylated forms.

In experiments carried out with a line of CHO cells adapted for growthin suspension culture in large tanks and or CHO cells adapted tosuspension culture in serum-free medium, no effect of manganese on thefraction of lower sialylated darbepoetin was observed.

Example 4

rHuEPO O-Site Characterization

CHO cells producing rHuEPO were grown as described in Example 1 with andwithout added 4 μM MgCl₂. Un-fractionated culture media collected afterthe third harvest cycle was analyzed for percent O-site occupancy ofrHuEPO. CHO cells producing darbepoetin were grown as described inExample 1 with and without added 4 μM MgCl₂. Conditioned media collectedafter each of the three harvest cycles was analyzed. CHO cells producingdarbepoetin were also grown as described in Example 1 with 0.4, 1, 4, 10and 40 μM MgCl₂ and media from the third harvest cycle was analyzed.

The percentage of O-linked sites occupied with glycosylation wasquantified by first removing N-linked structures by N-Glycanase (Sigma)digestion of the cell culture media, followed by reverse phase HPLCchromatography. Specifically, 5 U of N-Glycanase (Sigma) was added to 10μL of concentrated (1:60) culture media samples and digested at 37° C.for three hours. The samples were then analyzed by reverse phasechromatography using a Zorbax C-8 column (150 mm×2.1 mm (VWR) using alinear AB gradient from 35%-60% B over 30 minutes (buffer A: 0.1% TFA inH₂O, buffer B: 0.1% TFA in 90% CH₃CN (Sigma)). The resultingchromatogram separates rHuEPO into two peaks; the first peak correspondsto the occupied O-site rHuEPO peptide while the smaller, second peakcorresponds to the unoccupied O-site rHuEPO peptide.

To confirm that these two peaks represented occupied and unoccupiedO-sites, fractions corresponding to these peaks were collected andcompared to N-Glycanase digested purified rHuEPO (Amgen Inc.). SDS-PAGEanalysis showed that N-Glycanase digestion reduces the apparentmolecular weight of rHuEPO from 32 kDal to a doublet with a majorcomponent of 18.5 kDal and a slightly faster migrating minor component.The larger peak migrated with the larger N-Glycanase digested rHuEPOband while the minor peak migrated with the smaller rHuEPO band. Lys-Cpeptide mapping in combination with mass spectrometry confirmed that thelarger peak corresponds to rHuEPO containing an O-linked carbohydratewhile the smaller peak corresponds to rHuEPO devoid of an O-linkedcarbohydrate.

Results of a representative experiment analyzing O-site occupancy forrHuEPO are displayed in FIG. 4 . FIG. 4 shows that the addition of 4 μMmanganese increased the rHuEPO O-site occupancy compared to control,from 88.1% to 91.6%.

Results of representative experiments for darbepoetin are displayed inFIGS. 5 and 6 . FIG. 5 shows data from each harvest cycle anddemonstrates that the addition of 4 μM manganese increased thedarbepoetin O-site occupancy compared to control, from 76.8% to 86%after the third harvest. FIG. 6 displays data from the third harvestcycle for darbepoetin and shows that O-site occupancy increased withincreasing concentrations of manganese. However, 40 μM manganeseadversely affected levels of protein production, resulting in a totalrelative decrease of 17% in the mg of darbepoetin produced over thethree harvest cycles combined. In contrast, 10 μM manganese did notappear to substantially affect protein production.

These data demonstrate that addition of Mn²⁺ to culture medium increasesO-site occupancy for erythropoietin and darbepoetin produced in CHO cellcultures.

Example 5

Evaluation of rHuEPO N-Site Branching and N-Site Occupancy

To determine N-site branching of rHuEPO or darbepoetin producedaccording to Example 1, the lower sialylated fractions are analyzed forstructural content by N-glycan mapping and enzymatic sequencing coupledwith MALDI-TOF mass spectrometry for size determination. Briefly, theprocedure calls for the N-glycans to be enzymatically released with 1U/mL N-Glycanase (Sigma), then desalted and deproteinated using PGCchromatography (VWR). The free N-glycans are labeled at the reducingterminus with a fluorescent tag 2-aminobenzamide (2AB) by reductiveamination (Sigma). A second cleanup procedure is performed utilizingpaper chromatography (VWR). The ^(2AB)N-glycan pools are mapped on aDionex PA1 column with fluorescence detection (excitation 330 nm,emission 420 nm) using a sodium acetate gradient of 50-150 mM at 1.67mM/min with sodium hydroxide (Sigma) at 50 mM isocratic. The^(2AB)N-glycan pools are desialylated using 1 U/mL A. ureafaciensneuraminidase with and without 0.5 U/mL B. testes galactosidase. Alldigests are performed at 37° C. for 18 h. Additional size analysis ofall the ^(2AB)N-glycan pools is obtained by MALDI-TOF mass spectrometry(Voyager, Applied BioSystems). The matrix is saturated 2,5-dihydroxybenzoic acid in 70% acetonitrile, 0.05% TFA (Sigma) and mixed at a 1:1ratio with the 2AB N-glycan pool on the probe. The MALDI settings are asfollows: Accelerating voltage: 20,000 V; Grid voltage: 94%; Guide wire:0.05%; Extraction delay time: 75 nsec; Laser intensity: 2300; Massrange: 500-5000.

To determine the N-linked occupancy of rHuEPO, 10 μg of a rHuEPOstandard (Amgen Inc.) is digested with 0.04 U of N-Glycanase (Sigma)overnight at room temperature to give a molecular weight ladder ofrHuEPO with varying N-linked occupied forms. 0.1 μg of total rHuEPOcollected from harvest media is loaded and separated by SDS-PAGE (Novex,Invitrogen) and then transferred to PVDF. Blots are probed with amonoclonal antibody to rHuEPO.

Example 6

Effect of Amino Acid Supplementation on rHuEPO Production andGlycosylation.

To determine the effect of addition of amino acids on production ofrHuEPO during cell culture, the levels of all twenty amino acids weredetermined by amino acid analysis in the starting media (“StandardMedia” of Example 1) and then again after five days of incubation in thethird harvest cycle as described in above in Example 1. Nine specificamino acids (non-essential and essential) were depleted to low levels(<3 mg/L) over this culture period: cysteine, isoleucine, leucine,tryptophan, valine, asparagine, aspartic acid, glutamate, and glutamine.The concentrations of these 8 amino acids in standard media were doubledto create enriched media. Enriched Medium consisted of serum-free 1:1DMEM/F-12 media supplemented with 1% amino acid stock (2.25 g/Lasparagine; 1.99 g/L aspartic acid; 1.76 g/L cysteine; 3.13 g/L cystine;2.21 g/L glutamic acid; 5.45 g/L isoleucine; 5.91 g/L leucine; 0.90 g/Ltryptophan; and 5.29 g/L valine). Cells were cultured in either StandardMedia or in Enriched Media, which was used throughout the entiretwenty-one day culture process. After the third harvest cycle, media wascollected and rHuEPO in the harvested media was quantitated by reversedphase HPLC.

CHO cells cultured in Enriched Media showed a modest increase of 12% inrHuEPO production compared to control cells cultured in Standard Media.However, although the Enriched Media improved rHuEPO protein production,the amount of lower sialylated material was increased two-fold, leadingto an overall decrease in highly sialylated rHuEPO as compared tocontrol cultures.

The change in the amount of rHuEPO found in the lower sialylated poolwas due to a lower degree of sialylation of the individual carbohydratesrather than a lower degree of carbohydrate branching. The structures ofN-linked carbohydrates found on rHuEPO in the lower sialylated fractionswere analyzed by MALDI-TOF. Results are displayed below in Table 1below.

TABLE 1 Results of MALDI-TOF analysis of rHuEPO N-Glycan lowersialylated pools. Observed Masses^(d) Control +Amino Acids +AminoAcids + Mn⁺⁺ Composition^(b) Theoretical AUN + AUN + AUN + Structure^(a)Hex HexN dHex Neu Mass^(c) None AUN Gal None AUN Gal None AUN Gal A 2 21 1036 1038 1038 1037 1039 1036 1038 B 3 2 1 1198 1200 1200 1198 C 2 3 11239 1240 1239 1238 1241 1238 1240 D 5 2 1376 1377 1379 1375 1378 13801374 1382 1375 1376 E 3 3 1 1401 1403 1402 1401 1403 1401 1402 1401 F 43 1 1563 1564 1562 1564 1562 1566 1562 G 3 4 1 1604 1605 1604 1604 16061604 1604 1604 H 4 4 1 1766 1766 1766 1767 1766 1769 1765 I 3 5 1 18071807 1806 1806 1809 1806 1807 1807 J 5 4 1 1928 1929 1927 1929 1927 19301927 K 4 5 1 1970 1970 1968 1970 1968 L 3 6 1 2011 2009 2011 2008 20092009 M 5 5 1 2132 2130 2129 2132 2129 N 4 6 1 2173 2172 2170 2172 2169 O5 4 1 1 2220 2221 P 6 5 1 2294 2292 2291 2293 2291 2291 Q 5 6 1 23352331 2331 2334 2332 R 6 6 1 2497 2495 2493 2495 2492 S 5 4 1 2 2511 2512T 7 6 1 2659 2656 2655 2657 2655 2657 ^(a)Structures A-T identified inFIG. 3 based on observed masses and general pathway of N-glycanbiosynthesis. ^(b)Hex, HexN, dHex and Neu indicate hexose,N-acetylhexosamine, deoxyhexose and N-acetyl neuraminic acid,respectively. ^(c)Theoretical masses are calculated based on the averagemass for the sodium adduct of a 2-aminobenzamide oligosaccharide withthe indicated composition ^(d)Observed masses for native (None),neuraminidase (AUN), and β-galactosidase (Gal) treated glycan's aregiven.

In order to confirm some of the masses seen by MALDI-TOF, the lowersialylated fraction was digested with either neuraminidase, orneuraminidase and galactosidase. Masses representing highly branchedcarbohydrate structures were analyzed and compared between the controlcultures grown in Standard Media and the cultures grown in the presenceof Enriched Media. Masses representing highly branched carbohydratestructures (FIG. 7 ; structures M-T) were observed. Amino acidsupplementation in the Enriched Media resulted in a mass correspondingto a highly branched structure that is missing all of its terminalgalactose (FIG. 7 , structure L). Similar masses of branched structuresmissing galactose were detected in both the control and Enriched Mediacultures (FIG. 7 , structures E, G-I, K, M-N, Q-R). These data indicatethat the degree of branching in rHuEPO produced in Standard Media andEnriched Media is similar and suggests that the loss of sialic acidresidues may be due to decreased galactosylation of highly branchedN-linked carbohydrates.

Example 7

Manganese Reversed Effects of Amino Acid Supplementation onGlycosylation

rHuEPO was cultured according to Example 1 in Enriched Media alone orEnriched Media plus 0.4, 4 and 40 μM MnCl₂. At early points in theculture; when cell numbers are low and the metabolic load is minimal,the Enriched Media has no effect on rHuEPO glycosylation. However,restoration of these depleted pools eventually caused defects in botholigosaccharide occupancy and sialylation, after the third harvestcycle.

Results of various representative experiments are shown in FIGS. 9, 10and 11 . FIG. 8 shows that culturing host cells in Enriched Media(supplemented with amino acids) reduced the percent of recoverablehighly sialylated rHuEPO (43%, control vs. 29%, with amino acidsupplementation). FIG. 8 also shows that adding manganese to theEnriched Media at 40, 4 and 0.4 μM improved percent recovery (31% at 40μM, 42% at 4 μM, 41% at 0.4 μM).

FIG. 9 shows that culturing host cells in Enriched Media (supplementedwith amino acids) increased the percent of lower sialylated rHuEPO formsobtained (7.3%, control vs. 13%, with amino acid supplementation). FIG.9 also shows that adding manganese to Enriched Media greatly reduced thepercent of lower sialylated rHuEPO produced at all concentrations ofMn²⁺ (1.6% at 40 μM, 2.1% at 4 μM, and 2.4% at 0.4 μM).

FIG. 10 shows that culturing host cells in Enriched Media (supplementedwith amino acids) reduces the percentage of O-site occupancy by sugarchains (76.2% control vs. 74.8% with amino acid supplementation). FIG.10 also shows that adding manganese to Enriched Media increased thepercentage occupancy of O-sites (84.4% at 40 μMm, 86.1% at 4 μM, 84.6%at 0.4 μM).

While the quality of rHuEPO was clearly improved by the addition of 40μM Mn²⁺ to cultures, the yield of rHuEPO protein secreted into the mediawas substantially reduced at this concentration of Mn²⁺ (to 36% ofcontrol at 40 μM Mn²⁺). Protein production levels remained high whenconcentrations of 4 and 0.4 μM Mn²⁺ were added to Enriched Media (109and 114% of control, respectively).

Additionally, the addition of Mn²⁺ resulted in masses consistent withbranched sugars containing fully galactosylated forms. Further digestionwith neuraminidase plus galactosidase confirmed these results as masseswere obtained consistent with core structures missing the galactoseresidues. Within the lower sialylated rHuEPO pool, the addition of Mn²⁺resulted in predominantly biantennary structures whereas the control andenriched amino acid media contained higher branched N-Glycans structuresmissing terminal galactose as described above (FIG. 7 ; structures H, J,O, S).

To rule out a possible limitation of the donor sugar nucleotide UDP-Galas a cause for reduced galactosylation each condition was also assayedfor relative quantities of UDP-Gal. The data show that the levels ofUDP-Gal in each condition were statistically indistinguishable andtherefore, not the cause for reduced glactosylation seen upon amino acidsupplementation in Enriched Media. Nor was UDP-Gal availability a factorin the improvement of galactosylation seen after Mn²⁺ addition.

Thus, the data show that Mn²⁺ addition to the Enriched Medium, at allconcentrations of Mn²⁺, markedly reduced the fraction of lowersialylated rHuEPO produced and increased recovery of highly sialylatedrHuEPO forms. The effects on sialylation were shown to increase withincreasing concentrations of manganese in a dose-dependent manner.Manganese addition also improved rHuEPO galactosylation in the lowersialylated fraction, and increased O-linked occupancy. The improvementin glycosylation was independent of the level of rHuEPO production.Evaluation of N-site occupancy by Western blot as described in Example 5also suggested that Mn²⁺ addition improved N-site occupancy.

All publications, patents and patent applications cited in thisspecification are herein incorporated by reference in their entirety,including but not limited to the material relevant for the reason cited,as if each individual publication or patent application werespecifically and individually indicated to be incorporated by reference.Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. A method for producing an erythropoietic composition comprisingsialylated erythropoiesis-stimulating molecules, comprising the stepsof: growing a manganese-responsive CHO host cell which produces anerythropoiesis-stimulating glycoprotein in a culture medium containingan amount of manganese effective to increase the sialylation of saiderythropoietic composition produced by said manganese-responsive hostcell, wherein the concentration of manganese in said culture mediumranges from about 0.01 to about 40 μM, and wherein theerythropoiesis-stimulating glycoproteins comprise (a) the amino acidsequence of SEQ ID NO: 3 (erythropoietin) or erythropoietic fragmentsthereof; or (b) the amino acid sequence of SEQ ID NO: 2 (darbepoetin) orerythropoietic fragments thereof.
 2. The method of claim 1, furthercomprising the step of recovering an erythropoietic composition whereinless than about 5% of the erythropoiesis-stimulating molecules are lowersialylated.
 3. The method of claim 1, in which the amount of manganeseis effective to increase the percentage of highly sialylatederythropoiesis-stimulating molecules.
 4. The method of claim 1, whereinthe amount of manganese is effective to increase the percentage oferythropoiesis-stimulating molecules which are glycosylated at potentialO-linked glycosylation sites.
 5. The method of claim 1, wherein theamount of manganese is effective to increase the percentage of galactoseamong the sugars attached to erythropoiesis-stimulating molecules. 6.The method of claim 1, wherein the culture medium is essentiallyserum-free. 7-10. (canceled)
 11. The method of claim 1, wherein themanganese is at a concentration of from about 0.4 to about 10 μM. 12.The method of claim 1, wherein the manganese is at a concentration offrom about 0.4 to about 4 μM.
 13. The method of claim 1, wherein theculture medium further comprises one or more supplementary amino acidsselected from the group consisting of asparagine, aspartic acid,cysteine, cystine, isoleucine, leucine, tryptophan, or valine.
 14. Themethod of claim 1, wherein the host cells are grown in roller bottles.15. The method of claim 1, wherein the manganese is added after a rapidcell growth phase.
 16. The method of claim 15, wherein the rapid cellgrowth phase lasts. for a period ranging between about 2 and 20 days.17. The method of claim 16, wherein the manganese is added after twoharvest cycles.
 18. The method of claim 17, wherein the first harvestcycle is about 8 days and the second harvest cycle is about 7 days long.19. A culture medium comprising CHO host cells producingerythropoiesis-stimulating glycoproteins and manganese at aconcentration from about 0.1 to about 40 μM, wherein theerythropoiesis-stimulating glycoproteins comprise (a) the amino acidsequence of SEQ ID NO: 3 (erythropoietin) or erythropoietic fragmentsthereof; or (b) the amino acid sequence of SEQ ID NO: 2 (darbepoetin) orerythropoietic fragments thereof.
 20. The culture medium of claim 19,wherein the host cells secrete an erythropoietic composition. 21.(canceled)
 22. The culture medium of claim 19 wherein the manganese isat a concentration of from about 0.1 to about 10 μM.
 23. The method ofclaim 1 further comprising the step of recovering the erythropoieticcomposition which comprises said sialylated erythropoiesis-stimulatingglycoproteins from the host cells and culture medium.
 24. The method ofclaim 1 further comprising the step of recovering the erythropoieticcomposition which comprises highly sialylated erythropoiesis-stimulatingglycoproteins from the host cells and culture medium.